Cephalosporin compounds

ABSTRACT

This invention provides cephalosporin compounds and salts thereof. Such compounds are useful for preparing cross-linked glycopeptide-cephalosporin antibiotics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/342,729, filed Jan. 30, 2006 now U.S. Pat. No. 7,332,471; whichapplication is a continuation of U.S. application Ser. No. 10/851,428,filed on May 21, 2004 (now U.S. Pat. No. 7,067,481 B2); whichapplication claims the benefit of U.S. Provisional Application No.60/473,065, filed on May 23, 2003; the entire disclosures of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to novel cross-linkedvancomycin-cephalosporin compounds which are useful as antibiotics. Thisinvention is also directed to pharmaceutical compositions comprisingsuch compounds; methods of using such compounds as antibacterial agents;and processes and intermediates for preparing such compounds.

2. State of the Art

Various classes of antibiotic compounds are known in the art including,for example, β-lactam antibiotics, such as cephalosporins, andglycopeptide antibiotics, such as vancomycin. Cross-linked antibioticcompounds are also known in the art. See, for example, U.S. Pat. No.5,693,791, issued to W. L. Truett and entitled “Antibiotics and Processfor Preparation”; WO 99/64049 A1, published on Dec. 16, 1999, andentitled “Novel Antibacterial Agents”. Additionally, WO 03/031449 A2,published on Apr. 17, 2003, and entitled “Cross-LinkedGlycopeptide-Cephalosporin Antibiotics” discloses compounds having aglycopeptide group covalently linked to the oxime moiety of acephalosporin group.

Due to the potential for bacteria to develop resistance to antibiotics,however, a need exists for new antibiotics having unique chemicalstructures. Additionally, a need exists for novel antibiotics havingimproved antibacterial properties including, by way of example,increased potency against Gram-positive bacteria. In particular, a needexists for new antibiotics that are highly effective againstantibiotic-resistant strains of bacteria, such as methicillin-resistantStaphylococci aureus (MRSA).

SUMMARY OF THE INVENTION

The present invention provides novel cross-linkedglycopeptide-cephalosporin compounds that are useful as antibiotics. Thecompounds of this invention have a unique chemical structure in which aglycopeptide group is covalently linked to a pyridinium moiety of acephalosporin group. Among other properties, compounds of this inventionhave been found to possess surprising and unexpected potency againstGram-positive bacteria including methicillin-resistant Staphylococciaureus (MRSA).

Accordingly, in one aspect, this invention provides a compound offormula I:

or a pharmaceutically acceptable salt thereof, wherein

each of X¹ and X² is independently hydrogen or chloro;

W is selected from the group consisting of N and CCl;

R² is hydrogen or C₁₋₆ alkyl;

one of R⁴ and R⁵ is hydroxy and the other is hydrogen;

each of R⁶ and R⁷ is independently hydrogen or methyl;

R⁸ is hydrogen or a group of the formula:

R⁹ is selected from hydrogen, C₁₋₆ alkyl, and C₃₋₆ cycloalkyl, wherealkyl and cycloalkyl may be substituted with —COOH or 1 to 3 fluorineatoms;

each R³ is independently selected from C₁₋₆ alkyl, —OR, halo, —SR,—S(O)R, —S(O)₂R, and —S(O)₂OR, where each R is independently C₁₋₆ alkyl,which may be substituted with COOH or 1 to 3 fluorine atoms;

n is 0, 1, 2 or 3;

x is 0, 1 or 2;

y is 0, 1 or 2;

R^(a) is —Y—R″—, where

R″ is selected from C₁₋₁₂ alkylene, C₂₋₁₂ alkenylene, C₂₋₁₂ alkynylene,C₃₋₆ cycloalkylene, C₆₋₁₀ arylene, C₂₋₉ heteroarylene, C₃₋₆ heterocycle,and combinations thereof, and is optionally substituted with 1 or 2groups selected from Z, where Z consists of —OR′, —SR′, —F, —Cl,—N(R′)₂, —OC(O)R′, —C(O)OR′, —NHC(O)R′, —C(O)N(R′)₂, —CF₃, and —OCF₃,and side chains of naturally occurring amino acids, where each R′ isindependently hydrogen or C₁₋₄ alkyl; and R″ contains at most 20non-hydrogen atoms;

Y, which links R″ to the pyridinium ring at a meta or para position, isselected from a direct bond, NR′, O (ether), S (sulfide), C(O)(carbonyl), NR′(CO), and (CO)NR′, precluding direct bonds betweenheteroatoms in Y and R″;

each R^(b) and R^(d) is independently selected from the group consistingof hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl;

each R^(c) is independently —Y′—R″—Y′—, where each Y′ is independentlyselected from the group consisting of a direct bond, O (ether), and NR′,precluding direct bonds between heteroatoms in Y′ and R″; and

each R^(e) is independently selected from the group defined by R″ above.

In another aspect, the invention provides a compound of formula II:

or a pharmaceutically acceptable salt thereof; wherein

W is selected from the group consisting of N and CCl;

R⁹ is selected from hydrogen, C₁₋₆ alkyl, and C₃₋₆ cycloalkyl, wherealkyl and cycloalkyl may be substituted with —COOH or 1 to 3 fluorineatoms;

the pyridinium ring has meta or para substitution;

R¹⁰ is hydrogen or C₁₋₆ alkyl; and

R¹¹ is C₁₋₁₂ alkylene.

In another of its composition aspects, this invention provides apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound of formulaI or formula II, or a pharmaceutically acceptable salt thereof,including any of the particular embodiments discussed herein.

Also provided are methods of inhibiting the growth of bacteria and/orinhibiting bacterial cell wall biosynthesis, by contacting bacteria witha growth-inhibiting amount of a compound of formula I or formula II, ora pharmaceutically acceptable salt thereof, including any of theparticular embodiments discussed herein. In particular, the methodsinclude those embodiments in which the compound is selected from thegroup consisting of those designated herein as Ia, Ib, Ic, Id, Ie, andIf.

In a related aspect, the invention provides a method of treating abacterial infection in a mammal, the method comprising administering toa mammal a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a therapeutically effective amount of a compoundof formula I or II, or a pharmaceutically acceptable salt thereof,including any of the particular embodiments discussed herein.

This invention is also directed to processes for preparing compounds offormula I or II or a salt thereof. Accordingly, in another of its methodaspects, this invention provides a process for preparing a compound offormula I or a salt thereof; the process comprising reacting a compoundof formula 1 or a salt, activated derivative, or protected derivativethereof, with a compound of formula 3 or 4 or a salt, activatedderivative, or protected derivative thereof, to provide a compound offormula I or a salt thereof; wherein the compounds of formula 1, 3 and 4are as defined herein.

Additionally, in another of its method aspects, this invention providesa process for preparing a compound of formula I or a salt thereof, theprocess comprising reacting a compound of formula 2 or a salt, activatedderivative, or protected derivative thereof; with a compound of formula5 or a salt, activated derivative, or protected derivative thereof; toprovide a compound of formula I or a salt thereof, wherein the compoundsof formula 2 and 5 are as defined herein.

In one embodiment, these processes further comprise the step of forminga pharmaceutically acceptable salt of a compound of formula I. Thisinvention is also directed to the product prepared by any of theseprocesses.

This invention is also directed to a compound of formula I or II, or apharmaceutically acceptable salt thereof, for use in therapy.Additionally, this invention is directed to the use of a compound offormula I or II, or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament, including a medicament for treating abacterial infection in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of cross-linked glycopeptide-cephalosporinantibiotics, in accordance with selected embodiments of the invention.

FIG. 2 shows a synthetic scheme for preparing cephalosporinintermediates that are useful for preparing cross-linkedglycopeptide-cephalosporin antibiotics.

FIGS. 3 to 6 show synthetic schemes for preparing representativecross-linked glycopeptide-cephalosporin antibiotics, designated hereinas Ia, Ib, Id, and Ie, respectively.

FIGS. 7 and 8 show synthetic schemes for preparing comparativedes-chloro analogs of cross-linked glycopeptide-cephalosporinantibiotics.

FIGS. 9 and 10 show synthetic schemes for preparing furtherrepresentative cross-linked glycopeptide-cephalosporin antibiotics.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides novel glycopeptide-cephalosporin compounds offormula I or II, or pharmaceutically acceptable salts thereof. Thesecompounds have multiple chiral centers and, in this regard, thecompounds are intended to have the stereochemistry shown. In particular,the glycopeptide portion of the compound is intended to have thestereochemistry of the corresponding naturally-occurring glycopeptide(i.e., vancomycin, chloroorienticin A and the like). The cephalosporinportion of the molecule is intended to have the stereochemistry of knowncephalosporin compounds. However, it will be understood by those skilledin the art that minor amounts of isomers having a differentstereochemistry from that shown may be present in the compositions ofthis invention provided that the utility of the composition as a wholeis not significantly diminished by the presence of such isomers.

Additionally, the linking portion of the compounds of this invention maycontain one or more chiral centers. Typically, this portion of themolecule will be prepared as a racemic mixture. If desired, however,pure stereoisomers (i.e., individual enantiomers or diastereomers) maybe used or a stereoisomer-enriched mixture can be employed. All suchstereoisomers and enriched mixtures are included within the scope ofthis invention.

In addition, compounds of this invention contain several acidic groups(i.e., carboxylic acid groups) and several basic groups (i.e., primaryand secondary amine groups) and therefore, the compounds of formula Ican exist in various salt forms. All such salt forms are included withinthe scope of this invention. Also, since the compounds of formula Icontain a pyridinium ring, an anionic counterion for the pyridiniumgroup may optionally be present including, but not limited to, halides,such as chloride; carboxylates, such as acetate; and the like.

Definitions

The following terms, as used herein, have the following meanings, unlessotherwise indicated:

The term “alkyl” refers to a monovalent saturated hydrocarbon groupwhich may be linear or branched. Unless otherwise defined, such alkylgroups typically contain from 1 to 10 carbon atoms. Representative alkylgroups include, by way of example, methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl,n-octyl, n-nonyl, n-decyl and the like.

The term “alkylene” refers to a divalent saturated hydrocarbon groupwhich may be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms. Representativealkylene groups include, by way of example, methylene, ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl,pentane-1,5-diyl and the like.

The term “alkenyl” refers to a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon double bonds. Unless otherwisedefined, such alkenyl groups typically contain from 2 to 10 carbonatoms. Representative alkenyl groups include, by way of example,ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl and thelike.

The term “alkynyl” refers to a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon triple bonds. Unless otherwisedefined, such alkynyl groups typically contain from 2 to 10 carbonatoms. Representative alkynyl groups include, by way of example,ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like.

The term “aryl” refers to a monovalent aromatic hydrocarbon having asingle ring (i.e., phenyl) or fused rings (i.e., naphthalene). Unlessotherwise defined, such aryl groups typically contain from 6 to 10carbon ring atoms. Representative aryl groups include, by way ofexample, phenyl and naphthalene-1-yl, naphthalene-2-yl, and the like.

The term “arylene” refers to a divalent aromatic hydrocarbon having asingle ring (i.e., phenylene) or fused rings (i.e., naphthalenediyl).Unless otherwise defined, such arylene groups typically contain from 6to 10 carbon ring atoms. Representative arylene groups include, by wayof example, 1,2-phenylene, 1,3-phenylene, 1,4-phenylene,naphthalene-1,5-diyl, naphthalene-2,7-diyl, and the like.

The term “cycloalkyl” refers to a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms. Representative cycloalkylgroups include, by way of example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and the like.

The term “cycloalkylene” refers to a divalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkylene groupstypically contain from 3 to 10 carbon atoms. Representativecycloalkylene groups include, by way of example, cyclopropane-1,2-diyl,cyclobutyl-1,2-diyl, cyclobutyl-1,3-diyl, cyclopentyl-1,2-diyl,cyclopentyl-1,3-diyl, cyclohexyl-1,2-diyl, cyclohexyl-1,3-diyl,cyclohexyl-1,4-diyl, and the like.

The term “halo” refers to fluoro, chloro, bromo and iodo.

The term “heteroaryl” refers to a monovalent aromatic group having asingle ring or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heteroaryl groups typicallycontain from 5 to 10 total ring atoms. Representative heteroaryl groupsinclude, by way of example, monovalent species of pyrrole, imidazole,thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole,isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine,indole, benzofuran, benzothiophene, benzimidazole, benzthiazole,quinoline, isoquinoline, quinazoline, quinoxaline and the like, wherethe point of attachment is at any available carbon or nitrogen ringatom.

The term “heteroarylene” refers to a divalent aromatic group having asingle ring or two fused rings and containing at least one heteroatom(typically 1 to 3 heteroatoms) selected from nitrogen, oxygen or sulfurin the ring. Unless otherwise defined, such heteroarylene groupstypically contain from 5 to 10 total ring atoms. Representativeheteroarylene groups include, by way of example, divalent species ofpyrrole, imidazole, thiazole, oxazole, furan, thiophene, triazole,pyrazole, isoxazole, isothiazole, pyridine, pyrazine, pyridazine,pyrimidine, triazine, indole, benzofuran, benzothiophene, benzimidazole,benzthiazole, quinoline, isoquinoline, quinazoline, quinoxaline and thelike, where the point of attachment is at any available carbon ornitrogen ring atom.

The term “heterocyclyl” or “heterocyclic” refers to a monovalent ordivalent saturated or unsaturated (non-aromatic) group having a singlering or multiple condensed rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heterocyclic groups typicallycontain from 2 to 9 total ring atoms. Representative heterocyclic groupsinclude, by way of example, monovalent species of pyrrolidine,imidazolidine, pyrazolidine, piperidine, 1,4-dioxane, morpholine,thiomorpholine, piperazine, 3-pyrroline and the like, where the point ofattachment is at any available carbon or nitrogen ring atom.

The term “cephalosporin” is used herein in its art-recognized manner torefer to a β-lactam ring system having the following general formula andnumbering system:

where R^(x) and R^(y) represent the remaining portion of thecephalosporin.

The term “glycopeptide antibiotic” or “glycopeptide” is used herein inits art-recognized manner to refer to the class of antibiotics known asglycopeptides or dalbahpeptides. See, for example, R. Nagarajan,“Glycopeptide Antibiotics”, Marcel Dekker, Inc. (1994) and referencescited therein. Representative glycopeptides include vancomycin, A82846A(eremomycin), A82846B (chloroorienticin A), A82846C, PA-42867-A(orienticin A), PA-42867-C, PA-42867-D and the like.

The term “vancomycin” is used herein in its art-recognized manner torefer to the glycopeptide antibiotic known as vancomycin. In thecompounds of the present invention, the point of attachment for thelinking moiety is at the “C-terminus” of vancomycin.

The term “cross-linked glycopeptide-cephalosporin antibiotics” refers tocovalent conjugation of a glycopeptide component to a cephalosporincomponent.

The term “pharmaceutically-acceptable salt” refers to a salt which isacceptable for administration to a patient, such as a mammal (e.g.,salts having acceptable mammalian safety for a given dosage regime).Such salts can be derived from pharmaceutically-acceptable inorganic ororganic bases and from pharmaceutically-acceptable inorganic or organicacids. Salts derived from pharmaceutically-acceptable inorganic basesinclude aluminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic, manganous, potassium, sodium, zinc and the like.Particularly preferred are ammonium, calcium, magnesium, potassium andsodium salts. Salts derived from pharmaceutically-acceptable organicbases include salts of primary, secondary and tertiary amines, includingsubstituted amines, cyclic amines, naturally-occurring amines and thelike, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically-acceptable acidsinclude acetic, ascorbic, benzenesulfonic, benzoic, camphosulfonic,citric, ethanesulfonic, fumaric, gluconic, glucoronic, glutamic,hippuric, hydrobromic, hydrochloric, isethionic, lactic, lactobionic,maleic, malic, mandelic, methanesulfonic, mucic, naphthalenesulfonic,nicotinic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric,tartaric, p-toluenesulfonic and the like. Particularly preferred arecitric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric andtartaric acids.

The term “salt thereof” refers to a compound formed when the hydrogen ofan acid is replaced by a cation, such as a metal cation or an organiccation and the like (e.g., an NH₄ ⁺ cation and the like). Preferably,the salt is a pharmaceutically acceptable salt, although this is notrequired for salts of intermediate compounds which are not intended foradministration to a patient.

The term “therapeutically effective amount” refers to an amountsufficient to effect treatment when administered to a patient in need oftreatment.

The term “treating” or “treatment” as used herein refers to the treatingor treatment of a disease or medical condition (such as a bacterialinfection) in a patient, such as a mammal (particularly a human or acompanion animal) which includes:

(a) preventing the disease or medical condition from occurring, i.e.,prophylactic treatment of a patient;

(b) ameliorating the disease or medical condition, i.e., eliminating orcausing regression of the disease or medical condition in a patient;

(c) suppressing the disease or medical condition, i.e., slowing orarresting the development of the disease or medical condition in apatient; or

(d) alleviating the symptoms of the disease or medical condition in apatient.

The term “growth-inhibiting amount” refers to an amount sufficient toinhibit the growth or reproduction of a microorganism or sufficient tocause death or lysis of the microorganism including gram-positivebacteria.

The term “cell wall biosynthesis-inhibiting amount” refers to an amountsufficient to inhibit cell wall biosynthesis in a microorganismincluding gram-positive bacteria.

The term “leaving group” refers to a functional group or atom which canbe displaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include chloro, bromo and iodogroups; and sulfonic ester groups, such as mesylate, tosylate,brosylate, nosylate and the like; activated ester groups, such as suchas 7-azabenzotriazole-1-oxy and the like; acyloxy groups, such asacetoxy, trifluoroacetoxy and the like.

The term “protected derivatives thereof” refers to a derivative of thespecified compound in which one or more functional groups of thecompound are protected from undesired reactions with a protecting orblocking group. Functional groups which may be protected include, by wayof example, carboxylic acid groups, amino groups, hydroxyl groups, thiolgroups, carbonyl groups and the like. Representative protecting groupsfor carboxylic acids include esters (such as a p-methoxybenzyl ester),amides and hydrazides; for amino groups, carbamates (such astert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters;for thiol groups, thioethers and thioesters; for carbonyl groups,acetals and ketals; and the like. Such protecting groups are well-knownto those skilled in the art and are described, for example, in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, New York, 1999, and references cited therein.

The term “amino-protecting group” refers to a protecting group suitablefor preventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to,tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), and the like.

The term “carboxy-protecting group” refers to a protecting groupsuitable for preventing undesired reactions at an carboxy group.Representative carboxy-protecting groups include, but are not limitedto, esters, such as methyl, ethyl, tert-butyl, benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), diphenylmethyl (benzhydryl, DPM) and thelike.

An “activated derivative”, with respect to a carboxylic acid orprotected derivative thereof, refers to the product, typically areactive ester, resulting from reaction of the carboxylic acid orderivative with an activating (coupling) agent, such as, for example,1-hydroxybenzotriazole (HOBT), 1-hydroxy-7-azabenzotriazole (HOAT), orothers described below or otherwise known in the art.

A “side chain of a naturally occurring amino acid” refers to the group Rin the formula HOOC—CHR—NH₂, where this formula represents an amino acidselected from alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine, and preferably selected from alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, isoleucine, leucine, lysine, methionine, serine, threonine, andvaline.

Representative Embodiments of Compounds of the Invention

The following substituents and values are intended to providerepresentative examples and embodiments of various aspects of thisinvention. These representative values are intended to further definesuch aspects and embodiments and are not intended to exclude otherembodiments or limit the scope of this invention. In this regard, therepresentation that a particular value or substituent is preferred isnot intended in any way to exclude other values or substituents fromthis invention unless specifically indicated.

In compounds of formula I, any heteroarylene or heterocyclic grouppresent in R″ preferably has 5 or 6, typically 6, total ring atoms, andeach aryl group has 6 total ring atoms. The group R″ is preferably analkylene chain, and is preferably linear.

Each group R³, when present, is preferably selected independently fromunsubstituted C₁₋₄ alkyl, unsubstituted C₁₋₄ alkoxy, fluoro, and chloro.In one embodiment, n is 1 or 2, and each R³ is independently selectedform methyl, methoxy, fluoro, and chloro. In another embodiment, n iszero, such that no group R³ is present.

In one preferred embodiment, R⁸ is hydrogen. In another preferredembodiment, R⁸ is a group of the formula:

In selected embodiments, R⁹ is hydrogen or C₁₋₄ alkyl, includinghydrogen or methyl. In one embodiment R⁹ is hydrogen. In anotherembodiment, R⁹ is methyl.

In one embodiment, W is CCl. In another embodiment, W is N.

Selected embodiments of other variables of formula I include,independently of each other: for X¹ and X², chloro; for R², hydrogen orC₁₋₄ alkyl; for R⁴ and R⁵, hydroxyl and hydrogen, respectively; for R⁶and R⁷, hydrogen and methyl, respectively; and for R⁸, hydrogen.

In other selected embodiments, R^(a) is —Y—R″—, where R″ is C₁₋₆alkylene, C₂₋₆ alkenylene, or C₂₋₆ alkynylene, and Y is selected from adirect bond, NR′, O, S, C(O), NR′(CO), and (CO)NR′, where R′ is hydrogenor methyl. In one embodiment, Y is a direct bond. In further embodimentsof the group R^(a), R″ is C₁₋₆ alkylene or, more preferably, C₁₋₄alkylene, e.g. methylene, ethylene, propylene or butylene.

Preferably, in the group R^(a), Y is a direct bond, and R″ is C₁₋₆alkylene or, more preferably, C₁₋₄ alkylene, e.g. methylene.

The pyridinium ring in formula I is typically meta or para substituted,more generally para substituted.

Preferably, the variable x is 0 or 1. When x is not 0, the group R^(b)is preferably selected from the group consisting of hydrogen, C₁₋₄ alkylor C₂₋₄ alkenyl. In one embodiment, R^(b) is hydrogen or C₁₋₄ alkyl.

The group R^(c), when x is not 0, is preferably —Y′—R″—Y′—, where eachY′ is independently selected from a direct bond, O, and NR′, where R′ ishydrogen or methyl, and R″ is selected from C₁₋₁₂ alkylene, C₂₋₁₂alkenylene, and C₂₋₁₂ alkynylene, wherein the alkylene, alkenylene oralkynylene groups are optionally substituted with 1 or 2 groups selectedfrom Z or the side chain of a naturally-occurring amino acid.Preferably, in the group R^(c), each Y′ is a direct bond, and R″ isC₁₋₁₂ alkylene or C₂₋₁₂ alkenylene, which may be substituted with 1 or 2groups selected from Z and a side chain of naturally-occurring aminoacid. In another embodiment of the group R^(c), R″ is C₁₋₁₂ alkylene,including C₁₋₆ alkylene, and is unsubstituted or substituted with a—COOH group.

Preferably, the variable y is 0 or 1. When y is not 0, the group R^(d)is preferably selected from the group consisting of hydrogen and C₁₋₄alkyl, and, in selected embodiments, hydrogen and methyl. In oneembodiment, R^(d) is H. The group R^(e), when y is not 0, is preferablyselected from C₁₋₁₂ alkylene, C₂₋₁₂ alkenylene, and C₂₋₁₂ alkynylene. Inone embodiment, R^(e) is C₁₋₁₂ alkylene. More preferably, R^(e) isselected from C₁₋₆ alkylene, C₂₋₆ alkenylene, and C₂₋₆ alkynylene, andmost preferably from C₂₋₄ alkenylene and C₁₋₄ alkylene.

The group R², in selected embodiments, is hydrogen or C₁₋₄ alkyl. Infurther embodiments, R² is hydrogen or methyl; in one embodiment, R² ishydrogen.

In selected embodiments, x and y are independently selected from 0and 1. Accordingly, preferred embodiments include compounds in whichx+y=0, compounds in which x+y=1, and compounds in which x+y=2.Alternatively, preferred embodiments include compounds in which the“linker” structure, represented by “L” in FIG. 1, includes no more thanabout 30 carbon atoms, excluding the pyridinium ring.

An example of a compound of the invention in which x=y=1 is the compounddesignated herein as Ia. Examples of invention compounds in which x=1and y=0 include those designated herein as Ib, Ic, and Ie. Examples ofinvention compounds in which x=y=0 include those designated herein as Idand If (see FIG. 1).

One exemplary class of compounds of structure I is that in which: x is 0or 1; y is 0 or 1; R^(a) is methylene; R^(b) (when x is 1) is hydrogen,methyl, or ethyl; R^(c) (when x is 1) is C₁₋₁₂ alkylene, preferably C₁₋₆alkylene, e.g. ethylene or n-pentylene (—(CH₂)₅—), which may besubstituted with —COOH; R^(d) (when y is 1) is hydrogen; and R^(e) (wheny is 1) is ethylene (—CH₂CH₂—). Specific embodiments of this class ofstructure I include the compounds designated herein as Ia, Ib, Ic, Id,Ie, and If. In one embodiment of this class, x is 1; y is 0; R^(a) ismethylene; R^(b) is hydrogen, methyl, or ethyl; and R^(c) is C₂₋₆alkylene, e.g. ethylene or n-pentylene (—(CH₂)₅—). Specific embodimentsof this class of structure I include the compounds designated herein asIb and Ic.

A subset of the invention compounds of formula I can also be defined byformula II. In selected embodiments of formula II, W is CCl. In otherselected embodiments, R⁹ is hydrogen or C₁₋₄ alkyl, including hydrogenor methyl.

R¹⁰ is hydrogen or C₁₋₆ alkyl, preferably hydrogen or C₁₋₄ alkyl,including hydrogen, methyl or ethyl. In another embodiment, R¹⁰ isethyl.

In further selected embodiments, R¹¹ is C₁₋₁₀ alkylene, preferably R¹¹is C₂₋₁₀ alkylene, and more preferably C₂₋₆ alkylene, e.g. —(CH₂)₂— or—(CH₂)₅—.

As for formula I above, the pyridinium ring in formula II is typicallymeta or para substituted, more generally para substituted.

Examples of invention compounds in accordance with formula II includethose designated herein as Ib and Ic (see FIG. 1).

While not intending to be limited by theory, the compounds of formulas Iand II are believed to inhibit bacterial cell wall biosynthesis, therebyinhibiting the growth of the bacteria or causing lysis of the bacteria.Accordingly, they are useful as antibiotics.

Among other properties, compounds of the invention have been found topossess surprising and unexpected potency against gram-positivebacteria, including methicillin-resistant Staphylococci aureus (MRSA)and methicillin-resistant Staphylococci epidermitis (MRSE), as describedfurther below.

General Synthetic Procedures

The cross-linked glycopeptide-cephalosporin compounds of this inventioncan be prepared from readily available starting materials, preferablyvia the intermediate compounds 1-5 described herein. It will beappreciated that where typical or preferred process conditions (i.e.,reaction temperatures, times, mole ratios of reactants, solvents,pressures, etc.) are given, other process conditions can also be used,as determined by one skilled in the art, unless otherwise stated.Optimum reaction conditions may vary with the particular reactants orsolvent used, but such conditions can be readily determined by oneskilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Suitableprotecting groups for a particular functional group, as well as suitableconditions for protection and deprotection of such functional groups,are well known in the art. Protecting groups other than thoseillustrated in the procedures described herein may be used, if desired.For example, numerous protecting groups, and means for theirintroduction and removal, are described in T. W. Greene and G. M. Wuts,Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York,1999, and references cited therein.

In a preferred method of synthesis, the compounds of formula I, where yis 0, or, in selected embodiments, the compounds of formula II, areprepared by reacting a glycopeptide of formula 1:

where R⁴-R⁸, X¹ and X² are as defined herein, or a salt, or an activatedC-terminal carboxyl derivative and/or amine-protected derivativethereof, with a compound of formula 3 or 4:

where W, R², R³, R⁹, R^(a-e), n, x, and y are as defined herein, or asalt or carboxy-protected derivative thereof; to provide a compound offormula I or II, or a salt or protected derivative thereof. Preferredembodiments of 1, 3 and 4 are as described above.

In preparing compounds of formula II, variables in structures 1, 3,and/or 4 are defined as follows: n is 0; x is 1; y is 0; R^(a) is CH₂;R^(b) is hydrogen or C₁₋₆ alkyl (as defined for R¹⁰ above); R^(c) isC₁₋₁₂ alkylene (as defined for R¹¹ above); R², R⁵, and R⁶ are hydrogen;R⁷ is CH₃; R⁹ is as defined herein; R⁴ is OH; and X¹ and X² are Cl.

Typically, the reaction is conducted by coupling glycopeptide 1, or asalt thereof, with about 0.5 to about 1.5 equivalents, preferably about0.9 to about 1.1 equivalents, of a compound of formula 3 or 4, in aninert diluent, such as DMF, using a conventional carboxylic acid-amine(peptide) coupling reagent, as discussed further below. In thisreaction, glycopeptide 1, or a salt thereof, is typically firstcontacted with the coupling reagent in the presence of an excess,preferably about 1.8 to about 2.2 equivalents, of an amine, such asdiisopropylethylamine at a temperature ranging from about −20° C. toabout 25° C., preferably at ambient temperature, for about 0.25 to about3 hours. Preferably, excess trifluoroacetic acid (typically about 2equivalents) is then added to form a TFA salt of any excessdiisopropylethylamine. The reaction is then generally cooled to atemperature of about −20° C. to about 10° C., preferably to about 0° C.,and intermediate 3 or 4 is added, followed by excess 2,4,6-collidine.This reaction is typically maintained at about 0° C. for about 1 toabout 6 hours, or until the reaction is substantially complete.

Alternatively, for preparing compounds of formula I where y is not 0, aglycopeptide derivative of formula 2, where R², R⁴-R⁸, R^(d-e), X¹ andX² are as defined herein, or a salt or protected derivative thereof, canbe reacted with intermediate of formula 5 where W, R³, R⁹, R^(a-c), nand x, are as defined herein, or a salt or activated or protectedderivative thereof. Preferred embodiments of 2 and 5 are as describedabove.

Typically, such a coupling reaction is carried out by reacting a salt ofintermediate 2, such as a trifluoroacetate salt, with about 0.5 to 1.5equivalents of an activated ester of intermediate 5, in the presence ofa base, such as collidine. Preferably, R^(d) is hydrogen in this case,so that reaction is favored at the C-terminal primary amine. See, forexample, the preparation of Ia illustrated in FIG. 3 and describedfurther in Example 4.

For preparation of compounds of formula I in which y>1, a similarstrategy can be followed, where one or more additions of an amino acid,e.g. of structure HNR^(d)—R^(e)—COOH, to the C-terminal —NHR^(d) groupof intermediate 2 precedes the reaction with intermediate 5.

Preferred coupling reagents, or activating reagents, for use in thesereactions include benzotriazol-1-yloxy tripyrrolidinophosphoniumhexafluorophosphate (PyBOP), preferably used in the amount of about 0.5to 1.5 equivalents, preferably about 0.9 to 1.1 equivalents, incombination with about 0.5 to 1.5 equivalents, preferably about 0.9 to1.1 equivalents, of 1-hydroxybenzotriazole (HOBT) or1-hydroxy-7-azabenzotriazole (HOAT). Other suitable coupling reagentsinclude O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU); bis(2-oxo-3-oxazolidinyl)phosphinic chloride(BOP-Cl); diphenylphosphoryl azide (DPPA); diphenylphosphinic chloride;diphenyl chlorophosphate (DPCP) and HOAT;1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC);pentafluorophenyl diphenylphosphinate, and the like.

After the coupling reaction is complete, any protecting groups presentin the product are then removed using conventional procedures andreagents. For example, deprotection of N-trityl,N-BOC(N-tert-butoxycarbonyl) and/or COO-PMB (para-methoxybenzyl ester)can be effected by treatment with excess trifluoroacetic acid and excessanisole or triethylsilane in an inert diluent, such as dichloromethaneor heptane, at ambient temperature for about 1 to about 12 hours, oruntil the reaction is complete. The deprotected product can be purifiedusing conventional procedures, such as column chromatography, HPLC,recrystallization and the like.

Glycopeptides of formula 1 suitable for use in the above procedure areeither commercially available or can be prepared by fermentation of theappropriate glycopeptide-producing organism, followed by isolation ofthe glycopeptide from the resulting fermentation broth using artrecognized procedures and equipment. The derivative 2 is readilyprepared by reaction of 1 with a diamine, as described, for example, inExample 3.

The cephalosporin intermediates 3 and 4 used in the above procedures arereadily prepared from commercially available starting materials andreagents using conventional procedures. By way of example, anintermediate of formula 4 can be prepared as shown in FIG. 2 anddescribed in Examples 1 and 2. Briefly,2-amino-5-chloro-α-methoxyimino-4-thiazole acetic acid (6) was reactedwith the amino cephalosporinic ester 7, catalyzed with EDAC, forming anamide linkage. This product (8 was then treated with TFA and anisole tocleave the PMB ester, followed by displacement of the primary chloridewith a pyridine derivative. The pyridine derivative containssubstituents —R^(a)—NHR² and optionally substituent(s) R³, as shown instructure 4 above. In the preparation shown in FIG. 2, the compound4-(N-t-BOC-N-ethyl)aminomethyl pyridine (9) is employed, such that R^(a)is methylene and R² is ethyl. This reaction gives the intermediate (10)in protected form; deprotection with TFA gives the intermediate 4a(intermediate 4 where W is CCl, R⁹ is Me, n is 0, R^(a) is CH₂, and R²is Et).

In an alternate process, the product 8 was reacted with sodium iodide inacetone, followed by reaction with 9. Reaction with TFA/anisole then wasused to remove both the Boc and PMB protecting groups.

Various substituted pyridines for use in the above reactions are eithercommercially available or can be prepared from commercially availablestarting materials and reagents using conventional procedures. Forexample, various aminoalkyl-substituted pyridines are commerciallyavailable, e.g. aminomethyl pyridines, where R^(a) is methylene, andaminoethyl pyridines, where R^(a) is ethylene, or can be prepared usingstandard organic synthesis procedures. Representative substitutedpyridine derivatives for use in this reaction include those in which R³is selected from methyl, methoxy, thiomethoxy, carboxythiomethoxy,fluoro, chloro, phenyl, cyclopropyl, carboxylic acid, carboxamide, andcombinations thereof. For preparation of compounds in which Y, whichlinks R″ to the pyridinium ring, is selected from NR′, O (ether), S(sulfide), C(O) (carbonyl), NR′(CO), and (CO)NR′), starting pyridinecompounds are commercially available or can be prepared by well knownprocedures. For example, 3-hydroxypyridine, 4-hydroxypyridine,3-aminopyridine, 4-aminopyridine, 4-mercaptopyridine, nicotinic acid andisonicotinic acid are commercially available from Aldrich Chemical Co,Milwaukee, Wis.

Intermediate 4 can be further substituted to form intermediates offormula 3. For example, FIG. 4 shows reaction of an intermediate offormula 4 with N-(t-BOC)-β-alanine (see Example 5, preparation of Ib)and with aspartic acid (see Example 8, preparation of Ie) to formintermediates of formula 3, where x is 1, after deprotection. Furtheraddition(s) of β-alanine or like compounds, such as other amino acids,could be employed to form intermediates of formula 3 in which x>1.

Reaction of intermediate 4 with a diacid, e.g. of the structureHOOC—R^(c)—COOH, can be employed (with suitable activating and/orprotecting reagents) to form intermediates of formula 5, in which x is 1(see FIG. 3; Example 4; preparation of Ia). To form intermediates offormula 5 in which x>1, intermediate 4 would first be reacted with oneor more moles of an amino acid (e.g. of the structureHOOC—R^(c)—NHR^(b)).

In preparing compounds in which, in the group R^(c), Y′ is selected fromO (ether) and NR′ (rather than a direct bond), linking moietiesincluding R^(c) will include one or more carbamate or urea linkages,rather than amide linkages. Such linkages can be formed by conventionalmethods. For example, an amine (such as —NHR² in intermediate 3 or 4 canbe reacted with an isocyanate or a chloroformate to form, respectively,a urea or carbamate linkage.

Further details regarding specific reaction conditions and proceduresfor preparing representative compounds of this invention orintermediates thereto are described in the Examples set forth below.

Pharmaceutical Compositions

The cross-linked glycopeptide-cephalosporin compounds of this inventionare typically administered to a patient in the form of a pharmaceuticalcomposition. Accordingly, in one of its composition aspects, thisinvention is directed to a pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient and a therapeuticallyeffective amount of a compound of formula I or II or a pharmaceuticallyacceptable salt thereof.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of this invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof bacterial infection. In this regard, the preparation of a suitablepharmaceutical composition for a particular mode of administration, suchas oral, topical, inhaled or parenteral administration, is well withinthe scope of those skilled in the pharmaceutical arts. Additionally, theingredients for such compositions are commercially available from, forexample, Sigma, P.O. Box 14508, St. Louis, Mo. 63178. By way of furtherillustration, conventional formulation techniques are described inRemington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia,Pa. 17^(th) Ed. (1985) and “Modern Pharmaceutics,” Marcel Dekker, Inc.3^(rd) Ed. (G. S. Banker & C. T. Rhodes, Eds.).

The pharmaceutical compositions of this invention will typically containa therapeutically effective amount of a compound of formula I or II or apharmaceutically acceptable salt thereof. Typically, such pharmaceuticalcompositions will contain from about 0.1 to about 90% by weight of theactive agent, and more generally from about 10 to about 30% of theactive agent.

Preferred pharmaceutical compositions of this invention are thosesuitable for parenteral administration, particularly intravenousadministration. Such pharmaceutical compositions typically comprise asterile, physiologically-acceptable aqueous solution containing atherapeutically effective amount of a compound of formula I or II or apharmaceutically-acceptable salt thereof.

Physiologically acceptable aqueous carrier solutions suitable forintravenous administration of active agents are well known in the art.Such aqueous solutions include, by way of example, 5% dextrose, Ringer'ssolutions (lactated Ringer's injection, lactated Ringer's plus 5%dextrose injection, acylated Ringer's injection), Normosol-M, Isolyte E,and the like.

Optionally, such aqueous solutions may contain a co-solvent, forexample, polyethylene glycol; a chelating agent, for example,ethylenediamine tetraacetic acid; a solubilizing agent, for example, acyclodextrin; an anti-oxidant, for example, sodium metabisulphite; andthe like.

If desired, the aqueous pharmaceutical compositions of this inventioncan be lyophilized and subsequently reconstituted with a suitablecarrier prior to administration. In a preferred embodiment, thepharmaceutical composition is a lyophilized composition comprising apharmaceutically-acceptable carrier and a therapeutically effectiveamount of a compound of formula I or II, or apharmaceutically-acceptable salt thereof. Preferably, the carrier inthis composition comprises sucrose, mannitol, dextrose, dextran, lactoseor a combination thereof. More preferably, the carrier comprisessucrose, mannitol, or a combination thereof.

In one embodiment, the pharmaceutical compositions of this inventioncontain a cyclodextrin. When used in the pharmaceutical compositions ofthis invention, the cyclodextrin is preferablyhydroxypropyl-β-cyclodextrin or sulfobutyl ether β-cyclodextrin. In suchformulations, the cyclodextrin will comprise about 1 to 25 weightpercent; preferably, about 2 to 10 weight percent of the formulation.Additionally, the weight ratio of cyclodextrin to active agent willtypically range from about 1:1 to about 10:1.

The pharmaceutical compositions of this invention are preferablypackaged in a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be packaged in sterile, hermetically-sealed ampoules and the like.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

Formulation Example A

A frozen solution suitable for preparing an injectable solution isprepared as follows:

Ingredients Amount Active Compound 10 to 1000 mg Excipients (e.g.,dextrose) 0 to 50 g Water for Injection Solution 10 to 100 mL

Representative Procedure: The excipients, if any, are dissolved in about80% of the water for injection and the active compound is added anddissolved. The pH is adjusted with 1 M sodium hydroxide to 3 to 4.5 andthe volume is then adjusted to 95% of the final volume with water forinjection. The pH is checked and adjusted, if necessary, and the volumeis adjusted to the final volume with water for injection. Theformulation is then sterile filtered through a 0.22 micron filter andplaced into a sterile vial under aseptic conditions. The vial is capped,labeled and stored frozen.

Formulation Example B

A lyophilized powder suitable for preparing an injectable solution isprepared as follows:

Ingredients Amount Active Compound 10 to 1000 mg Excipients (e.g.,mannitol and/or sucrose) 0 to 50 g Buffer Agent (e.g., citrate) 0 to 500mg Water for Injection 10 to 100 mL

Representative Procedure: The excipients and/or buffering agents, ifany, are dissolved in about 60% of the water for injection. The activecompound is added and dissolved and the pH is adjusted with 1 M sodiumhydroxide to 3 to 4.5 and the volume is adjusted to 95% of the finalvolume with water for injection. The pH is checked and adjusted, ifnecessary, and the volume is adjusted to the final volume with water forinjection. The formulation is then sterile filtered through a 0.22micron filter and placed into a sterile vial under aseptic conditions.The formulation is then freeze-dried using an appropriate lyophilizationcycle. The vial is capped (optionally under partial vacuum or drynitrogen), labeled and stored under refrigeration.

Formulation Example C

An injectable solution for intravenous administration to a patient isprepared from Formulation Example B above as follows:

Representative Procedure: The lyophilized powder of Formulation ExampleB (e.g., containing 10 to 1000 mg of active compound) is reconstitutedwith 20 mL of sterile water and the resulting solution is furtherdiluted with 80 mL of sterile saline in a 100 mL infusion bag. Thediluted solution is then administered to the patient intravenously over30 to 120 minutes.

Utility

The cross-linked glycopeptide-cephalosporin compounds of the inventionare useful as antibiotics. For example, the compounds of this inventionare useful for treating or preventing bacterial infections and otherbacteria-related medical conditions in mammals, including humans andtheir companion animals (i.e., dogs, cats, etc.) that are caused bymicroorganisms susceptible to the compounds of this invention.

Accordingly, in one of its method aspects, the invention provides amethod of treating a bacterial infection in a mammal, the methodcomprising administering to a mammal in need of such treatment, apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a therapeutically effective amount of a compound of formulaI or II, or a pharmaceutically-acceptable salt thereof.

By way of illustration, the compounds of this invention are particularlyuseful for treating or preventing infections caused by Gram-positivebacteria and related microorganisms. For example, the compounds of thisinvention are effective for treating or preventing infections caused bycertain Enterococcus spp.; Staphylococcus spp., including coagulasenegative staphylococci (CNS); Streptococcus spp.; Listeria spp.;Clostridium ssp.; Bacillus spp.; and the like. Examples of bacterialspecies effectively treated with the compounds of this inventioninclude, but are not limited to, methicillin-resistant Staphylococcusaureus (MRSA); methicillin-susceptible Staphylococcus aureus (MSSA);glycopeptide intermediate-susceptible Staphylococcus aureus (GISA);methicillin-resistant Staphylococcus epidermitis (MRSE);methicillin-sensitive Staphylococcus epidermitis (MSSE);vancomycin-sensitive Enterococcus faecalis (EFSVS); vancomycin-sensitiveEnterococcus faecium (EFMVS); penicillin-resistant Streptococcuspneumoniae (PRSP); Streptococcus pyogenes; and the like.

As shown in Table 2 of Example 16 below, several invention compounds,Ia-f, Im and In, were more effective than vancomycin againstmethicillin-sensitive Staphylococcus aureus and methicillin-resistantStaphylococcus aureus, by a factor of 10 or more. The compounds werealso significantly more active than their des-chloro analogs Ig, Ij, andIk, although these compounds were also more active than vancomycin. In a“time-kill” assay, as described in Example 17, a compound of formula I,i.e. compound Ib, was bactericidal against MRSA at a concentration of≦1.0 μg/mL in 4 hours. By comparison, vancomycin was bactericidalagainst MRSA at a concentration of 4 μg/mL in 24 hours. In an in vivoassay in neutropenic mice, as described in Example 18, a compound offormula I, i.e. compound Ib, had an ED₅₀ of <0.1 mg/kg, iv, compared toan ED₅₀ of 9 mg/kg, iv, for vancomycin.

In general, the compounds of the invention are preferred for treating orpreventing infections caused by strains of bacteria that are susceptibleto either glycopeptides or cephalosporins.

Representative types of infections or bacteria-related medicalconditions which can be treated or prevented with the compounds of thisinvention include, but are not limited to, skin and skin structureinfections, urinary tract infections, pneumonia, endocarditis,catheter-related blood stream infections, osteomyelitis, and the like.In treating such conditions, the patient may already be infected withthe microorganism to be treated or merely be susceptible to infection inwhich case the active agent is administered prophylactically.

The compounds of this invention are typically administered in atherapeutically effective amount by any acceptable route ofadministration. Preferably, the compounds are administered parenterally.The compounds may be administered in a single daily dose or in multipledoses per day. The treatment regimen may require administration overextended periods of time, for example, for several days or for one tosix weeks or longer. The amount of active agent administered per dose orthe total amount administered will typically be determined by thepatient's physician and will depend on such factors as the nature andseverity of the infection, the age and general health of the patient,the tolerance of the patient to the active agent, the microorganism(s)causing the infection, the route of administration and the like.

In general, suitable doses will range of from about 0.25 to about 10.0mg/kg/day of active agent, preferably from about 0.5 to about 2mg/kg/day. For an average 70 kg human, this would amount to about 15 toabout 700 mg per day of active agent, or preferably about 35 to about150 mg per day.

Additionally, the compounds of this invention are effective forinhibiting the growth of bacteria. In this embodiment, bacteria arecontacted either in vitro or in vivo with a growth-inhibiting amount ofa compound of formula I or II or pharmaceutically acceptable saltthereof. Typically, a growth-inhibiting amount will range from about0.008 μg/mL to about 50 μg/mL; preferably from about 0.008 μg/mL toabout 25 μg/mL; and more preferably, from about 0.008 μg/mL to about 10μg/mL. Inhibition of bacterial growth is typically evidenced by adecrease or lack of reproduction by the bacteria and/or by lysis of thebacteria, i.e., by a decrease in colony-forming units in a given volume(i.e., per mL) over a given period of time (i.e., per hour) compared tountreated bacteria.

The compounds of this invention are also effective for inhibiting cellwall biosynthesis in bacteria. In this embodiment, bacterial arecontacted either in vitro or in vivo with a cell wallbiosynthesis-inhibiting amount of a compound of formula I or II orpharmaceutically acceptable salt thereof. Typically, a cell wallbiosynthesis-inhibiting amount will range from about 0.04 μg/mL to about50 μg/mL; preferably from about 0.04 μg/mL to about 25 μg/mL; and morepreferably, from about 0.04 μg/mL to about 10 μg/mL. Inhibition of cellwall biosynthesis in bacteria is typically evidenced by inhibition orlack of growth of the bacteria including lysis of the bacteria.

Additionally, compounds of this invention have been found to havesurprising and unexpectedly rapid cidality against certain bacteria,including methicillin-resistant Staphylococci aureus (MRSA) andmethicillin-resistant Staphylococci epidermitis (MRSE). Theseproperties, as well as the antibiotic utility of the compounds of thisinvention, can be demonstrated using various in vitro and in vivo assayswell known to those skilled in the art. For example, representativeassays are described in further detail in the following Examples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention.

In the examples below, the following abbreviations have the followingmeanings unless otherwise indicated. Abbreviations not defined belowhave their generally accepted meaning.

BOC=tert-butoxycarbonyl

CFU=colony-forming units

DCM=dichloromethane

DIPEA=diisopropylethylamine

DMF=N,N-dimethylformamide

DMSO=dimethyl sulfoxide

EtOAc=ethyl acetate

HOBT=1-hydroxy benzotriazole

HOAT=1-hydroxy-7-azabenzotriazole

HPLC=high performance liquid chromatography

MIC=minimum inhibitory concentration

MS=mass spectrometry

PMB=p-methoxybenzyl

PyBOP=benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate

THF=tetrahydrofuran

TLC=thin layer chromatography

TFA=trifluoroacetic acid

All temperatures reported in the following examples are in degreesCelsius (° C.) unless otherwise indicated. Also, unless noted otherwise,reagents, starting materials and solvents were purchased from commercialsuppliers (such as Aldrich, Fluka, Sigma and the like) and were usedwithout further purification. Vancomycin hydrochloride semi-hydrate waspurchased from Alpharma, Inc., Fort Lee, N.J. 07024 (Alpharma AS, Oslo,Norway).

Reverse-phase HPLC was typically conducted using a C₁₈ column and (A)98% water, 2% acetonitrile, 0.1% TFA, with an increasing gradient (e.g.,0 to about 70%) of (B) 10% water, 90% acetonitrile, 0.1% TFA, unlessotherwise stated.

In the syntheses described below, intermediates 4a-4g are defined asfollows. In each of these compounds, n is 0 and R^(a) is —CH₂—.

Cmpd. W R⁹ R^(a) subst. R² 4a C—Cl CH₃ para CH₂CH₃ 4b C—Cl CH₃ para H 4cC—Cl CH₃ meta H 4d C—H CH₃ para H 4e C—H CH₃ meta H 4f N CH₃ para H 4gC—Cl H para H

Example 1 Preparation of 4a:(7R)-7-[(Z)-2-(2-Amino-5-chlorothiazol-4-yl)-2-(methoxyimino)acetamido]-3-[((4-(ethylamino)methyl)-1-pyridinio)methyl]-3-cephem-4-carboxylateBis-trifluoroacetic Acid Salt (see FIGS. 2 and 3) A. Preparation of2-Amino-5-chloro-α-(methoxyimino)-4-thiazoleacetic Acid (6)

To 500 mL of acetic acid were added 40.0 g (0.20 mol) of2-amino-α-(methoxyimino)-4-thiazoleacetic acid (Aldrich ChemicalCompany, Milwaukee, Wis.) and 27.9 g (0.21 mol) N-chlorosuccinimide. Themixture was heated in a 70° C. bath. The solids dissolved within 30minutes, and after 45 minutes the reaction was complete by MS. The darksolution was allowed to cool to room temperature and concentrated undervacuum to give a dark solid, which was used without purification.

B. Preparation of Cephalosporin Derivative (8)

To 47.5 g (0.20 mol) of (6) in 700 mL of DMF was added 81.1 g (0.20 mol)of the aminocephalosporonic ester (7) (Otsuka, Osaka, JP) and 27.0 g(0.20 mol) of HOAt. The mixture was cooled to 0° C., and 26.7 g (0.22mol) of 2,4,6-collidine was added. To this solution was added 42.2 g(0.22 mol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDAC). After five hours, the solution was partitionedbetween 1:1 EtOAc/ether and water, and the phases were separated. Theaqueous solution was back-extracted twice with EtOAc. The combinedorganic phases were washed once each with 0.5 M citric acid, water, 50%saturated NaHCO₃, water, and brine. The organic phase was dried overMgSO₄, filtered, and concentrated in vacuo. The residue was dissolved ina minimum volume of methylene chloride, and the product was precipitatedby pouring the solution into ether. The solids were isolated byfiltration to give 48 g of the compound (8).

Analytical Data: MS m/z calc. 586.04, obs. 586.2 (M+1); ¹H NMR(DMSO-d₆): d 9.60 (d, 1H), 7.35 (m, 3H), 6.91 (d, 2H), 5.82 (m, 1H),5.17 (m, 3H), 4.56 (m, 2H), 3.84 (s, 3H), 3.76 (s, 3H), 3.62 (m, 2H).

C. Coupling to Pyridinium Moiety

To 48.5 g (82.7 mmol) of the chloromethylcephalosporin ester 8 and 40 mLof anisole in 400 mL of 1,2-dichloroethane was added 300 mL of TFA.After 40 minutes, the solution was concentrated to ½ volume in vacuo,and the product was precipitated by pouring the concentrate into 1 L ofether. The solids were isolated by filtration, rinsed with ether, anddried to give 42.5 g of crude free acid. This material (25.0 g, 53.6mmol) was dissolved in 200 mL of 1:1 acetonitrile/DMF. To this solutionwere added 10.1 g (83.3 mmol) of 2,4,6-collidine and 15.2 g (64.5 mmol)of 4-(N-t-butoxycarbonyl)-ethylaminomethyl pyridine (9) (prepared byconventional procedures from 4-ethylaminomethylpyridine, which iscommercially available from Aldrich Chemical Company, Milwaukee, Wis.).After 3.5 hours, the product was precipitated by pouring the solutioninto 1 L of ether. The solids were isolated by filtration, washed withether, and dried under vacuum to give 26 g of crude material. Theproduct was purified by preparative reversed-phase HPLC to give compound10 as an off-white powder.

Analytical Data: MS m/z calc. 666.12, obs. 666.2 (M+1); ¹H NMR(DMSO-d₆): d 9.36 (d, 1H), 8.91 (d, 2H), 7.95 (d, 2H), 7.24 (b, 1H),5.83 (m, 1H), 5.50 (m, 2H), 5.17 (d, 1H), 4.66 (s, 2H), 3.83 (s, 3H),3.52 (d, 1H), 3.30 (m, 3H), 1.37 (s, 9H), 1.08 (t, 3H).

D. Deprotection of 10

To 610 mg (0.78 mmol) of compound 10 and 1 mL of anisole was added 8 mLof trifluoroacetic acid. After 20 minutes, the product was precipitatedby pouring the reaction into 100 mL of ether. The solids were isolatedby filtration, washed with ether, and dried under vacuum to give 4a as alight yellow solid.

Analytical Data: ¹H NMR (DMSO-d₆): d 9.56 (d, 1H), 9.44 (br, 1H), 9.10(d, 2H), 8.21 (d, 2H), 7.38 (b, 1H), 5.86 (m, 1H), 5.54 (m, 2H), 5.15(d, 1H), 4.51 (s, 2H), 3.80 (s, 3H), 3.40 (m, 2H), 3.30 (m, 2H), 1.22(t, 3H).

Example 2 Preparation of 4b:(7R)-7-[(Z)-2-(2-Amino-5-chlorothiazol-4-yl)-2-(methoxyimino)acetamido]3-[(4-(aminomethyl)-1-pyridinio)methyl]-3-cephem-4-carboxylateBis-trifluoroacetic Acid Salt A. Preparation of2-Amino-5-chloro-α-(methoxyimino)-4-thiazoleacetic acid (6)

To 500 mL of DMF were added 50.0 g (250 mmol) of2-amino-α-(methoxyimino)-4-thiazoleacetic acid and 35 g (260 mmol) ofN-chlorosuccinimide. The mixture was stirred at room temperatureovernight, after which time mass spectral analysis showed no morestarting material to be present. The light brown solution was usedwithout further manipulation.

B. Preparation of Cephalosporin Derivative (8)

To the solution of the acid 6 in DMF (101.5 g, 250 mmol) from step (a)was added the aminocephalosporonic ester 7 (34 g, 250 mmol). The mixturewas cooled to 0° C., and 33.5 mL (250 mmol) of 2,4,6-collidine wasadded. To this solution was added 53 g (275 mmol) of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After 2hours, the solution was precipitated into 3 L water and filtered. Thesolids were washed with water (2×1 L), saturated sodium bicarbonate (500mL) and water (4×500 mL) and dried under vacuum. The dried solids weretaken up in 500 mL methylene chloride at room temperature, and thesolution was slowly stirred, forming a precipitate. The crystals werecollected by filtration, washed with methylene chloride until thewashings were no longer brown, and dried under vacuum to give the amide8 (74 g).

C. Coupling to Pyridinium Moiety

Acetone (250 mL) was added to a mixture of 50 g (85 mmol)chloromethyl-cephalosporin ester 8 and 13 g (85 mmol) sodium iodide,under nitrogen in the dark. After stirring for 30 minutes, 27 g (130mmol) of 4-(N-tert-butoxycarbonyl)aminomethyl pyridine and 30 mL acetonewere added. After stirring an additional 2 hours, 1.4 L of 0.1 N HCl wasadded, producing a gummy precipitate. The solvent portion was decanted,and the gummy residue was treated with 800 mL water to give a solid. Thewater was decanted, and the solid was dissolved in 1 L of 4:1 ethylacetate/ethanol. The solution was washed with 500 mL saturated brine,dried over magnesium sulfate, and evaporated to dryness to give 70 g (79mmol, 93%) of the product (analogous to 10, with —NHBOC substituted for—NEtBOC) was obtained, having a purity of 78% as determined by HPLC (254nm).

This material was deprotected, without further purification, as follows.The crude product (70 g, 79 mmol) was dissolved in 550 mL methylenechloride under nitrogen, and 35 mL (320 mmol) anisole was added,followed by 150 mL trifluoroacetic acid. After 2 hours, the mixture wasconcentrated under vacuum. The product precipitated on addition of 1 Ldiethyl ether. The solids were isolated by filtration, washed withether, stirred in 200 mL water, and filtered. The filtrate waslyophilized to dryness and purified by reverse-phase HPLC, yielding 30 g(approx. 50%) 4b.

Example 3 Preparation of 2a: C-Aminoethylamide Vancomycin (Structure 2where R², R⁵, R⁶, R⁸ are H; R⁴ is OH; R⁷ is Me; X¹, X² are Cl; R^(d) isH; R^(e) is —CH₂CH₂—)

To a solution of vancomycin hydrochloride monohydrate (7.3 g, 4.7 mmol)in 75 mL of DMSO was added, at room temperature, 4.1 mL (23.5 mmol)DIPEA, followed by 1.8 g (5.6 mmol) 9-fluorenylmethylN-(2-aminoethyl)carbamate hydrochloride (Fmoc-NH—CH₂—CH₂—NH₃Cl) (seeFIG. 3). A solution of PyBOP (2.7 g, 5.2 mmol) and HOBT (0.63 g, 4.7mmol) in 75 mL of N,N′-dimethylpropyleneurea (DMPU) was then addeddropwise rapidly. The resulting solution was stirred at room temperaturefor 2 hrs, then poured into 800 mL of diethyl ether to give a gum. Theether was decanted and the gum washed with additional ether to givecrude [C]-(2-Fmoc-aminoethyl) vancomycin.

This product (Fmoc-2a) was taken up in 40 mL of DMF and 10 mL ofpiperidine was added, and the solution was left to stand at roomtemperature for 20 minutes, then added dropwise to 450 mL acetonitrile,forming a precipitate. The mixture was centrifuged, the acetonitriledecanted, and the residue washed twice with 450 mL of acetonitrile andonce with 450 mL of diethyl ether and then air dried. The residue wasthen taken up in water, acidified to pH<5 with 3N HCl, and purified byreverse-phase HPLC, using a gradient of 2 to 30% acetonitrile in watercontaining 0.1% trifluoroacetic acid. This gave the product 2a as thetri(TFA) salt.

Example 4 Preparation of Compound Ia

Pyridinium lactam bis-trifluoroacetate 4a (2.4 g), prepared as describedin Example 1 above, was dissolved in N,N-dimethylformamide (DMF, 40 mL)under nitrogen and cooled to 0° C. Dodecanedioic acidbis-1-hydroxy-7-azatriazole ester (7.0 g) was added (11), followed by2,4,6-collidine (1.2 mL), and the mixture was stirred for 65 minutes,then added to ethyl acetate (50 mL), and this mixture precipitated intodiethyl ether (400 mL). The activated ester lactam 5a was collected byfiltration and dried under vacuum, and a portion (14 mg) was dissolvedin DMF (122 μL) and added at 0° C. to a mixture of C-aminoethylamidevancomycin tritrifluoroacetate, 2a (50 mg), prepared as described inExample 3, and 2,4,6-collidine (5.4 μL) in DMF (500 μL). The mixture waskept at 0° C. for 20 minutes, then trifluoroacetic acid was added (7.3μL). The mixture was kept at −20° C. overnight, then purified byreverse-phase HPLC to give the product Ia.

Analytical Data: MS m/z obs. 2252.8, calc. 2252.7.

Example 5 Preparation of Compound Ib

At room temperature under nitrogen, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (740 mg), 1-hydroxy-7-azatriazole (520 mg)and N-(tert-butoxycarbonyl)-β-alanine (660 mg) were stirred in DMF (10mL) for 7 hours, then cooled to 0° C. Pyridinium lactambis-trifluoroacetate 4a (2 g) (see Example 1) was added and stirreduntil it had fully dissolved, then 2,4,6-collidine (700 μL) was added,and the mixture was stirred at 0° C. for 100 minutes. Trifluoroaceticacid (700 μL) and water (30 mL) were added, and the product was purifiedby reverse-phase HPLC. After lyophilization, the product was treatedwith 20 mL of 50% trifluoroacetic acid/dichloromethane for 40 minutes.The lactam amine 3a (see FIG. 4) was recovered as itsbis-trifluoroacetate salt on precipitation into diethyl ether, and driedunder vacuum.

Vancomycin hydrochloride monohydrate, 1a (900 mg) (Structure 1 where R⁵,R⁶, R⁸ are H; R⁴ is OH; R⁷ is Me; X¹, X² are Cl; also referred to inFigures as “vancomycin”) was dissolved in dimethyl sulfoxide (5 mL)under nitrogen at room temperature, and 1-hydroxy-7-azatriazole wasadded (80 mg), followed by PyBOP (300 mg) in DMF (5 mL) andN,N-diisopropylethylamine (100 μL). The mixture was stirred for 20minutes, trifluoroacetic acid (45 μL) was added, and the mixture wascooled to 0° C. Pyridinium lactam amine 3a (250 mg) was added, followedby 2,4,6-collidine (270 μL), and the mixture was stirred at 0° C. for 30minutes, then precipitated into acetonitrile. The crude product wascollected by centrifugation and purified by reverse-phase HPLC to giveIb.

Analytical Data: MS m/z obs. 2069.7, calc. 2069.4.

Example 6 Preparation of Compound Ic

This compound was prepared according to the procedure described inExample 5, substituting 6-(N-BOC)amino hexanoic acid forN-BOC-β-alanine.

Analytical Data: MS m/z obs. 2110.5, calc. 2111.5

Example 7 Preparation of Compound Id

Vancomycin hydrochloride monohydrate, 1a (900 mg), was dissolved indimethyl sulfoxide (5 mL) under nitrogen at room temperature, and1-hydroxy-7-azatriazole was added (80 mg), followed by PyBOP (300 mg) inDMF (5 mL) and NAN-diisopropylethylamine (100 μL). The mixture wasstirred for 20 minutes, trifluoroacetic acid (45 μL) was added, and themixture was cooled to 0° C. Pyridinium lactam bis-trifluoroacetate 4c(250 mg) (identical to 4b with the exception of meta substitution onpyridine; see FIG. 5) was added, followed by 2,4,6-collidine (270 μL),and the mixture was stirred at 0° C. for 30 minutes, then precipitatedinto acetonitrile. The crude product was collected by centrifugation andpurified by reverse-phase HPLC to give the product Id.

Analytical Data: MS m/z obs. 1970.9, calc. 1970.3.

Example 8 Preparation of Compound Ie A. Preparation of 3b (structure 3where W is Cl, R⁹ is Me, n is 0, R^(a) is CH₂, R^(b) is hydrogen, R^(c)is —CH₂CH(COOH)—, R² is hydrogen, and x is 1)

HOAT (56 mg, 0.2 mmol), PyBOP (208 mg, 0.2 mmol) and N—BOC aspartic acidα-tert-butyl ester was dissolved in DMF (500 μL). DIPEA (70 μL, 0.2mmol) was then added, and the reaction was stirred at room temperaturefor 20 minutes. The reaction was then cooled to −10° C., and TFA (32 μL,0.2 mmol) was added. The C3-pyridinium cephalosporin 4b (306 mg, 0.2mmol) was added to the reaction as a solution in DMF (500 μL). Collidine(160 μL, 0.6 mmol) was added and the reaction was stirred for 1 hr at−10° C. The reaction mixture was concentrated in vacuo, then purifiedusing reverse phase HPLC (10-90% gradient over 60 minutes) to provide,on lyophilization, the protected cephalosporin aspartic acid derivativeTFA salt as a white amorphous powder (193 mg). This product (162 mg) wasdissolved in TFA (10 mL) and stirred at room temperature for 60 minutes.The reaction was concentrated in vacuo, and the residue was dissolved inCH₃CN/H₂O (1:1; 0.1% TFA) and lyophilized to provide the desireddeprotected cephalosporin aspartic acid derivative (3b) TFA salt as awhite solid (92 mg).

Analytical Data: MS m/z 652.9 (MH⁺).

B. Reaction of 3b with Vancomycin

Vancomycin hydrochloride, 1a (0.32 g, 0.21 mmol), was dissolved in 3 mLof DMSO. A 0.5 mL solution of PyBOP (0.11 g, 0.21 mmol) and HOAT (0.03g, 0.21 mmol) in DMF was added, followed by DIPEA (0.04 mL, 0.21 mmol).The reaction was stirred 30 minutes, then TFA (0.02 mL, 0.21 mmol) wasadded, and the mixture was cooled to 0° C. A solution of 3b (0.15 g,0.17 mmol) (see FIG. 6) in DMF (1 mL) was then added, followed bycollidine (0.08 mL, 0.63 mmol). The reaction was stirred at 4° C.overnight and then precipitated from EtOAc, centrifuged and theresulting solid washed with MeCN. The desired compound was purified byHPLC (2-30% MeCN) to give 0.3 g of Ie as a white solid.

Analytical Data: MS m/z 1042.6 [(M+2H)/2)]²⁺.

Example 9 Preparation of Compound If

To a solution of 0.6 mL of DMSO containing vancomycin hydrochloride, 1a(134.0 mg, 0.09 mmol), and HOAT (12.3 mg, 0.09 mmol) was added asolution of PyBOP (46.9 mg, 0.09 mmol) in 0.6 mL of DMF, followed byaddition of diisopropylethylamine (31.4 μL, 0.2 mmol). After stirring atambient temperature for 20 min, the reaction mixture was treated withTFA (13.9 μL, 0.2 mmol) while cooling at 0° C. To this activated esterof vancomycin was added 69 mg (0.09 mmol) of pyridinium lactam 4a andcollidine (51.3 μL, 0.4 mmol). The final mixture was allowed to stir atroom temperature for 3 h, prior to quenching with TFA (33.4 μL, 0.40mmol). The crude product was precipitated from the reaction mixture byadding ethyl acetate. The solid was collected by spinning down, dried,and purified by preparative HPLC. The desired product If was obtained asa white fluffy solid (89 mg).

Analytical Data: Retention time (anal. HPLC: 10 to 70% MeCN/H₂O over 6min)=2.00 min. MS m/z calcd. 1970.27 (C₈₆H₉₄Cl₃N₁₆O₂₈S₂); obsd. 985.7[(M+2H)/2]²⁺.

Example 10 Preparation of Compound Ik (a des-chloro analog of CompoundIf)

Sodium iodide (0.271 g, 1.81 mmol) was added to a solution ofcephalosporin-β-lactam 12 (0.999 g, 1.81 mmol) (see FIG. 7) dissolved inacetone (25 mL), protected from light and stirred under nitrogen. After25 minutes, 4-(N-BOC-aminomethyl)pyridine (0.390 g, 1.87 mmol) wasadded, and the reaction was stirred for 2 hours. The product wasprecipitated with a large excess of ethyl acetate and recovered byfiltration.

The product (0.353 g, 0.488 mmol) was dissolved in DCM (2.5 mL) andanisole (0.21 mL, 1.93 mmol) and TFA (1.5 mL, 19.47 mmol) were added.The reaction mixture was then stirred for 45 minutes at room temperatureunder nitrogen. The product was precipitated using diethyl ether,centrifuged, and the pellet washed twice with diethyl ether and dried.The product was dissolved in water and insolubles were filtered off. Theaqueous material was lyophilized to afford 0.166 g of the TFA salt of 4das an orange solid (identical to 4b with the exception that W=CH).

Analytical Data: MS m/z 504.1 (M+1); HPLC (2-30 acetonitrile/water, 254nm) retention time=0.270 min.

HOAt (0.0626 g, 0.460 mmol) was added to a solution of vancomycinhydrochloride (0.7078 g, 0.455 mmol) in DMSO (3.5 mL). PyBOP (0.2338 g,0.449 mmol) dissolved in DMF (3.5 mL) and DIPEA (79.0 μL, 0.453 mmol)were added, and the reaction was stirred at room temperature undernitrogen for 20 minutes, followed by addition of TFA (35.0 μL, 0.454mmol). The reaction was then cooled in an ice bath. Lactam amine 4d(0.166 g, 0.227 mmol) was added, and upon dissolution collidine (180.0μL, 1.365 mmol) was added. After 50 minutes of stirring, TFA (125.0 μL,1.62 mmol) was added, and the product was precipitated with acetonitrileand centrifuged. The pellet was redissolved in a minimal amount of DMFand reprecipitated with acetonitrile three times, then dried undervacuum to give 0.8 g crude product. The crude product was purified byprep HPLC (2-35 acetonitrile/water), and fractions containing compoundIk were collected and lyophilized.

Analytical Data: MS m/z 1539.6 (fragment), HPLC: (2-30acetonitrile/water, 254 nm) retention time=2.92 min.

Example 11 Preparation of Compound Ig (a des-chloro analog of CompoundIb) A. Preparation of N-Ethyl-N-(4-pyridylmethyl)-3-(N-BOC)-aminoPropanamide (13)

To a solution of N-t-BOC-β-alanine (4.678 g, 24.7 mmol) in DCM (50 mL)was added diisopropylcarbodiimide (3.87 g, 24.7 mmol). The mixture wascooled in an ice bath and 4-(ethylaminomethyl)pyridine (3.37 g) wasadded. The reaction mixture was then stirred for 3.5 hours. The solidwas removed by filtration, and the filtrate was washed with water anddried under vacuum to give 5.34 g of the title intermediate.

Analytical Data: MS m/z 308.1 (M+1).

B. Preparation of Compound Ig

Sodium iodide (0.485 g, 3.22 mmol) was added to a solution ofcephalosporin-β-lactam 12 (2.031 g, 3.22 mmol) (see FIG. 8) dissolved inacetone (50 mL) protected from light and under nitrogen. After 20minutes, 13, prepared as described above, (1.29 g, 3.40 mmol) in acetone(10 mL) was added, and the reaction was stirred for 6 hours. The productwas precipitated with a large excess of ethyl acetate and recovered byfiltration. This product (1.70 g, 1.90 mmol) was dissolved in DCM (5 mL)and anisole (0.825 mL, 7.6 mmol) and TFA (5 mL, 64.9 mmol) were added.The reaction mixture was then stirred for 1 hour at room temperatureunder nitrogen. The product was precipitated using diethyl ether,centrifuged and the pellet washed twice with diethyl ether and dried, togive 1.7 g of the TFA salt of 3c (identical to 3a with the exceptionthat W is CH).

Analytical Data: MS m/z 603.3 (M+1); HPLC (2-90 acetonitrile/water, 254nm) retention time=3.42 min.

HOAt (0.560 g, 4.11 mmol) was added to a solution of vancomycinhydrochloride (6.350 g, 4.08 mmol) in DMSO (30 mL). PyBOP (2.127 g, 4.09mmol) dissolved in DMF (30 mL) and DIPEA (0.711 mL, 4.08 mmol) wereadded, and the reaction was stirred at room temperature under nitrogenfor 20 minutes, followed by addition of TFA (0.314 mL, 4.08 mmol). Thereaction was then cooled in an ice bath. Lactam amine 3c (1.7 g, 2.04mmol) dissolved in DMSO (25 mL) and DMF (10 mL) was added, and upondissolution collidine (1.89 mL, 14.3 mmol) was added. The reaction wasstirred for 50 minutes, TFA (1.26 mL, 16.3 mmol) was added, and theproduct was precipitated with acetonitrile and centrifuged down. Thepellet was redissolved in a minimal amount of DMF and reprecipitatedwith acetonitrile three times, then dried under vacuum to give 7.4 gcrude product. This crude product was purified by preparative HPLC (2-35acetonitrile/water), and fractions containing the product, Ig, werecollected and lyophilized.

Analytical Data: MS m/z 1639.5 (fragment), HPLC: (2-30acetonitrile/water, 254 nm) retention time=3.76 min.

Example 12 Preparation of Compound Ij (a des-chloro analog of CompoundId)

Sodium iodide (1.4 g, 9.1 mmol) was added to(7R)-7-[(Z)-2-(2-amino-thiazol-4-yl)-2-(methoxyimino)acetamido]-3-(chloromethyl)-3-cephem-4-carboxylate(5 g, 9.1 mmol) in 35 mL of acetone. The reaction vessel was wrapped infoil and the mixture stirred for 1 hour. A solution of3-aminomethylpyridine (2.8 g, 13.8 mmol) in 5 mL of acetone was added,and the reaction was stirred for 70 minutes and then precipitated intoether to give 5.4 g crude yield of 4e (identical to 4c with theexception that W is CH). This solid (4.4 g) was suspended in 20 mL ofDCM, and 20 mL of TFA was added. The reaction mixture was stirred atroom temperature for 90 minutes and then concentrated. The resulting oilwas precipitated from ether and purified by preparative HPLC (2-10%MeCN) to give 0.6 g of the TFA salt of 4e, as a white fluffy solid.

Analytical Data: R_(t)=0.8 min (10-70% MeCN). ¹H NMR (DMSO-d₆): δ 3.25(dd, 2H), 3.8 (s, 3H), 4.2 (bs, 2H), 5.05 (d, 1H), 5.45 (dd, 2H), 5.8(dd, 1H), 7.05 (s, 1H), 8.2 (m, 1H), 8.45 (bs, 2H), 8.6 (d, 1H), 9.0 (d,1H), 9.05 (s, 1H), 9.4 (dd, 1H).

Vancomycin hydrochloride (0.73 g, 0.49 mmol) was dissolved in 2.5 mL ofDMSO. A solution of PyBOP (0.23 g, 0.45 mmol) and HOAT (0.06 g, 0.45mmol) was added in 2 mL of DMF, followed by DIPEA (0.16 mL, 0.82 mmol).The reaction was stirred for 25 minutes, then TFA (0.07 mL, 8.2 mmol)was added, and the reaction was cooled to 0° C. Collidine was added(0.22 mL, 1.6 mmol), followed by a solution of the above lactam 4e in 2mL of DMF. The reaction was stirred for 2 h, then precipitated fromether and centrifuged. The product was purified by HPLC and thenlyophilized to yield 0.23 g of the product, Ij.

Example 13 Preparation of Compound Im

2-Amino-α-(methoxyimino)-4-thiadiazoleacetic acid (15) (4.0 g, 19.8mmol), prepared according to J. Antibiotics 53(10): 1061 (2000), wascombined with cephalosporin derivative 14, prepared according to Bull.Chem. Soc. Jpn. 43:2925-33 (1970) (8.42 g, 20.79 mmol) (see FIG. 9) andHOAT (3.33 g, 21.78 mmol) in 50 mL DMF. The solution was purged withnitrogen and cooled to 0° C., and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (3.99 g, 20.79 mmol) wasadded, followed by 1,3,5-collidine (2.75 mL, 20.79 mmol). The solutionwas stirred at 0° C. for 2 hours, then the reaction mixture was pouredinto 300 mL of water. The resulting solid was filtered, triturated withsaturated aqueous NaHCO₃, refiltered, washed twice with water, andair-dried overnight to yield the cephalosporin intermediate 16 as anoff-white powder. This compound was used without further purification.

Analytical Data: MS m/z calc. 553.01; obsd. 553.0 (M⁺, ³⁵Cl), 555.0 (M⁺,³⁷Cl).

The chloro compound 16 (4.0 g, 7.23 mmol) was placed in a flask with4-t-BOC-aminomethylpyridine (2.26 g, 10.85 mmol) and sodium iodide (1.08g, 7.23 mmol) and purged with N₂. Acetone (60 mL) was added, and thereaction mixture was stirred at room temperature for 2 hours, thenpoured into 300 mL of 0.2 M HCl, resulting in the formation of a red gumon the sides of the flask. The acetone was decanted and the gum wasdried under vacuum. Once dry, the residue was dissolved in 20 mL of DCM,and TFA (20 mL) was added. The reaction was stirred for one hour afterwhich LC/MS analysis indicated the deprotection was complete. Thissolution was then poured into 200 mL of Et₂O, and the resultingprecipitate was filtered, washed with Et₂O and dried under vacuum. Oncedry, the crude solid was triturated in 100 mL of water for 3 hours. Thissolution was filtered, and the product was lyophilized to yield thecrude compound 4f (identical to 4b with the exception that W is N) as abis-TFA salt. The compound was used without further purification.

Analytical Data: MS m/z calc. 491.0; obsd. 491.5

Vancomycin hydrochloride (0.2 g, 0.135 mmol) and HOAt (20.6 mg, 0.135mmol) were dissolved in 2 mL of DMSO. To this solution was added asolution of PyBOP (70.2 mg, 0.0135 mmol) in 2 mL of DMF. DIPEA (23.5 μL,0.135 mmol) was added, and the solution was stirred at room temperaturefor 20 min. After this time, a solution of 4f (49 mg, 0.0675 mmol) in 1mL of DMF was added, and the solution was cooled to 0° C.1,3,5-Collidine (62.5 μL, 0.473 mmol) was then added, and the solutionwas stirred at 0° C. for 45 min. TFA (150 μL) was then added, and thesolution was poured into 70 mL of Et₂O. The resulting precipitate wasfiltered, washed with Et₂O and dried under vacuum. The product waspurified by reverse-phase HPLC and isolated by lyophilization to affordIm as the tri-TFA salt.

Analytical Data: MS m/z calc. 1937.8; obsd. 1937.6.

Example 14 Preparation of Compound In

To a stirred solution of 2-amino-α-(trityloxyimino)-4-thiazole aceticacid (17) (15.60 g, 36.3 mmol) (prepared by tritylation ofethyl-2-amino-α-(hydroxyimino)-4-thiazoleacetate, followed byhydrolysis) in DMF (100 mL) was added 4.85 g (36.3 mmol)N-chlorosuccinimide (see FIG. 10). The reaction mixture was stirred atroom temperature for 15 h, then poured into water (500 mL), and theprecipitated solid was filtered and dried to yield the 5-chloro compound(15.11 g, 90%) as an off-white solid.

Analytical Data: ¹H NMR (DMSO-d, 300 MHz): δ 7.18-7.33 (m, 15H); MS m/zobsd. 486.4 [M+Na]⁺.

To a stirred solution of this compound (28.0 g, 60 mmol) in 250 mL ofDMF were added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (12.7 g, 66mmol), HOBT (9.0 g, 66 mmol) and 2,4,6-collidine (8.8 mL, 66 mmol). Thereaction mixture was cooled to 0° C., and after 5 min 14 (see Example13; FIG. 9) (24.4 g, 60 mmol) was added. After 3.5 h, the reactionmixture was diluted with ethyl acetate (1200 mL) and washed with 1.0MHCl (250 mL), satd. aqueous sodium bicarbonate (250 mL) and brine (250mL). The organic layer was dried (MgSO₄), filtered and concentrated invacuo to yield the cephalosporin intermediate 18 (43.2 g, 88%) as anoff-white solid, which was used without further purification.

Analytical Data: ¹H NMR (DMSO-d₆, 300 MHz): δ 3.65 (q, 2H), 3.76 (s,3H), 4.54 (q, 2H), 5.24 (s, 2H), 5.27 (d, 1H), 6.03 (q, 1H), 6.95 (d,2H), 7.18-7.41 (m, 19H), 9.90 (s, 1H).

Sodium iodide (920 mg, 61 mmol) and N-t-BOC-4-(aminomethyl)pyridine (1.8g, 86 mmol) were added to a stirred solution of 18 (5 g) in acetone (20mL) in the dark (foil wrapped flask). The reaction mixture was stirredat room temperature for 2.5 h, then added portionwise to diethyl ether(200 mL) and the resulting solid collected by filtration. A solution ofthe crude solid (5.9 g) in TFA/DCM (40 mL, 1:1) was stirred at roomtemperature for 1 h, then added portionwise to diethyl ether (300 mL).The resulting solid was collected by filtration to afford a solidresidue which was purified by preparative HPLC to yield the TFA salts oftwo hydroxyimino stereoisomers (240 mg and 120 mg, respectively) of 4g(identical to 4b with the exception that R⁹ is hydrogen), each as awhite solid.

Analytical Data:

Major stereoisomer of 4g: ¹H NMR (DMSO-d₆, 300 MHz): δ 3.40 (q, 2H),4.45 (s, 2H), 5.18 (d, 1H), 5.54 (q, 2H), 5.87 (q, 1H), 7.31 (bs, 2H),8.18 (d, 2H), 8.71 (bs, 3H), 9.09 (d, 2H), 9.39 (d, 1H), 11.73 (s, 1H);MS m/z obsd. 524.2 [M+H]⁺.

Minor stereoisomer of 4g: ¹H NMR (DMSO-d₆, 300 MHz): δ 3.50 (q, 2H),4.45 (s, 2H), 5.19 (d, 1H), 5.58 (q, 2H), 5.83 (q, 1H), 7.27 (bs, 2H),8.20 (d, 2H), 8.77 (bs, 3H), 8.81 (d, 1H), 9.10 (d, 2H), 12.51 (s, 1H);MS m/z obsd. 524.2 [M+H]⁺.

A solution of PyBOP (170 mg, 0.33 mmol) and HOAt (44 mg, 0.33 mmol) inDMF (1 mL) was added to a stirred solution of vancomycin hydrochloride(485 mg, 0.33 mmol) in DMSO/DMF (6 mL, 3:1). DIPEA (0.057 mL, 0.33 mmol)was subsequently added, and the reaction mixture was stirred at roomtemperature for 30 min. TFA (0.026 mL, 0.33 mmol) was added to thereaction mixture, which was then cooled to 0° C., and collidine (0.097mL, 0.73 mmol) and a solution of 4 (123 mg, 0.16 mmol) in DMF (1 mL)were then added. The reaction mixture was allowed to warm to roomtemperature over 4 h, then added portionwise to ethyl acetate (50 mL),and the resulting solid was collected by filtration and purified bypreparative HPLC to yield the TFA salt of the product In (174 mg, 46%)as a white solid.

Analytical Data: MS m/z obsd. 979.4 [(M-pyridine)/2]⁺.

Example 15 Determination of Aqueous Solubility

The aqueous solubility of compounds of the invention was determinedusing the following procedure. A 5 wt. % dextrose buffer solution at pH2.2 was prepared by adding 1 mL of 1 N hydrochloric acid (Aldrich) to 99mL of a 5 wt. % aqueous dextrose solution (Baxter). A 1 mg/mL stocksolution for calibration standards was then prepared by dissolving 1 mgof the test compound in 1 mL of DMSO. This solution was vortexed for 30seconds and then sonicated for 10 minutes. The stock solution was thendiluted with water to prepare calibration standards having the followingconcentrations: 50, 125, 250, 375 and 500 μg/mL.

Each test compound (30 mg) was weighed into a Millipore non-sterile,Ultrafree-MC 0.1 μm filter unit (Millipore UFC30VVOO) and a magneticstir bar was added to each unit. The 5 wt. % dextrose buffer solution(750 μL) was then added to each unit and these mixtures were vortexedfor 5 minutes. The filter units were then placed in an Eppendorf tuberack and the tube rack was placed on top of a magnetic stirrer. Eachunit was then titrated to pH 3 using 1 N NaOH (VWR) and the resultingsolutions centrifuged at 7000 rpm for 5 minutes. Each unit was thendiluted 200 fold with 5% dextrose buffer solution and the dilutedsamples were transferred into auto sampler vials for analysis.

The calibration standards and the test samples were analyzed byreverse-phase HPLC using the following conditions:

Column: Luna 150×4.6 mm; C18; 5μ

Mobile phase: A=5/95, B=95/5, both=MeCN/H₂O; 0.1% TFA

Method: 10 m Lido 100 (0-100% B in 6 min)

Injection volume: 20 μL

Wavelength: 214 nm

The solubility of each test sample was calculated by comparing the peakarea of the test sample to the calibration curve and multiplying by thedilution factor.

TABLE 1 Solubility (mg/mL) in 5% aqueous dextrose buffer Compound No.Solubility Ib 9.63 Id 10.0 Ie 10.0 If 5.49

Example 16 Determination of Minimal Inhibitory Concentrations (MICs)

Minimal inhibitory concentration (MICs) assays were performed using thebroth microdilution method set forth in NCCLS guidelines (see, NCCLS.2000. Methods for Dilution Antimicrobial Susceptibility Tests forBacteria That Grow Aerobically; Approved Standard-Fifth Ed., Vol. 20,No. 2). Bacterial strains were obtained from the American Type TissueCulture Collection (ATCC), Stanford University Hospital (SU), KaiserPermanente Regional Laboratory in Berkeley (KPB), Massachusetts GeneralHospital (MGH), the Centers for Disease Control (CDC), the San FranciscoVeterans' Administration Hospital (SFVA) or the University of CaliforniaSan Francisco Hospital (UCSF). Vancomycin-resistant enterococci werephenotyped as Van A or Van B based on their sensitivity to teicoplanin.Some vancomycin-resistant enterococci that had been genotyped as Van A,Van B, Van C1 or Van C2 were also obtained from the Mayo Clinic.

In this assay, cryopreserved bacterial cultures of reference andclinical strains were streaked for isolation on appropriate agar medium(i.e., Trypticase Soy Agar, Trypticase Soy Agar with defibrinated sheeperythrocytes, Brain Heart Infusion Agar, Chocolate Agar). Followingincubation to allow formation of colonies, these plates were sealed withParafilm® and stored refrigerated for up to two weeks. For preparationof assay inocula and to ensure low variability, several colonies from abacterial isolate cultured on the agar plates were pricked with aninoculating loop and aseptically transferred to Mueller-Hinton Broth(supplemented with divalent cations to required levels based onmanufacturer's certification). The broth culture was grown overnight at35° C., diluted in fresh prewarmed broth and grown to log phase; this isequivalent to a 0.5 MacFarland standard or 1×10⁸ colony forming unitsper milliliter (CFU/mL). Not all cell suspensions, due to speciesvariability, contained 1×10⁸ CFU/mL when turbidity is equivalent to theMacFarland standard, therefore acceptable adjustments (based on NCCLSguidelines) were made in dilutions of different bacterial strains. Theinoculum was diluted such that 100 μL of this culture in Mueller-HintonBroth, supplemented Mueller-Hinton Broth, or Haemophilus test medium,when over layered onto a 2-fold serially diluted series of antibioticconcentrations also in 100 μL of corresponding medium, in a 96-wellmicrotiter plate resulted in a starting bacterial concentration of 5×10⁵CFU/mL. The plates were then incubated 18-24 hours at 35° C. The MIC wasread visually as the lowest concentration well with no bacterial growth.Bacterial growth is defined as more than three pinpoint colonies, abutton of precipitated cells larger than 2 mm in diameter, or obviousturbidity.

Strains routinely tested in the initial screen includedmethicillin-sensitive Staphylococcus aureus (MSSA),methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcusaureus producing penicillinase, methicillin-sensitive Staphylococcusepidermidis (MSSE), methicillin-resistant Staphylococcus epidermidis(MRSE), vancomycin-sensitive Enterococcus faecium (EFMVS),vancomycin-sensitive Enterococcus faecalis (EFSVS), vancomycin-resistantEnterococcus faecium also resistant to teicoplanin (EFMVR Van A),vancomycin-resistant Enterococcus faecium sensitive to teicoplanin(EFMVR Van B), vancomycin-resistant Enterococcus faecalis also resistantto teicoplanin (EFSVR Van A), vancomycin-resistant Enterococcus faecalissensitive to teicoplanin (EFSVR Van B), penicillin-sensitiveStreptococcus pneumoniae (PSSP) and penicillin-resistant Streptococcuspneumoniae (PSRP). Because of the inability of PSSP and PSRP to growwell in Mueller-Hinton broth, MICs with those strains were determinedusing either TS broth supplemented with defibrinated blood orHaemophilus test medium.

Test compounds having significant activity against the strains mentionedabove were then tested for MIC values in a larger panel of clinicalisolates including the species listed above as well as non-speciatedcoagulase negative Staphylococcus both sensitive and resistant tomethicillin (MS-CNS and MR-CNS). Additionally, these test compounds werealso assayed for MICs against gram-negative microorganisms, such asEscherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae,Enterobacter cloacae, Acinetobacter baumannii, Haemophilus influenzaeand Moraxella catarrhalis.

Table 2 shows MIC₉₀ data for a compound of this invention againstmethicillin-resistant S. aureus (MRSA) and methicillin-susceptible S.aureus (MSSA) as compared to the known antibiotic, vancomycin.

TABLE 2 Minimum Inhibitory Concentrations (MICs), in μg/mL Compound MIC(μg/mL) MRSA 33591 MSSA 13709 Ia 0.13 <0.1 Ib <0.1 <0.1 Ic <0.1 <0.1 Id<0.1 <0.1 Ie <0.1 <0.1 If <0.1 <0.1 Ig 0.78 <0.1 Ij 0.78 <0.1 Ik 0.45<0.1 Im <0.1 <0.1 In <0.1 <0.1 Vancomycin 2.0 1.0

The data in Table 2 demonstrate that compounds of this invention (i.e.,Ia, Ib, Ic, Id, Ie, If, Im and In) had surprising and unexpectedantibacterial activity against MRSA 33591 compared to either thedes-chloro analogs Ig, Ij and Ik or vancomycin; and that compounds ofthis invention had surprising and unexpected antibacterial activityagainst MSSA 13709 compared to vancomycin.

Example 17 Time-Kill Assay

This time-kill assay is a method for measuring the rate of bactericidalactivity of a test compound. These procedures are similar to thosedescribed in V. Lorian, “Antibiotics in Laboratory Medicine”, FourthEdition, Williams and Wilkins (1996), pages 104-105. A rapid time-killis desirable to quickly prevent bacterial colonization and reduce hosttissue damage.

Bacterial inocula were prepared as described in Example 16 fordetermination of MIC. Bacteria were diluted in prewarmed media in shakeflasks and incubated with shaking (200 rpm, 35° C.). At 0, 1, 4, and 24hours samples were withdrawn from the flasks and bacteria wereenumerated by plate counting. Subsequent to the initial sampling, acompound to be assayed was added to the shake flask culture. Platecounts at these intervals previous to and following addition of thecompound were expressed graphically in a time-kill curve. Bactericidalactivity is defined as a ≧3 log decrease (reduction greater than orequal to 99.9%) in bacterial cell numbers by 24 hours.

In this assay, a compound of formula I, i.e. compound Ib, wasbactericidal against MRSA 33591 at a concentration of ≦1.0 μg/mL in 4hours. By comparison, vancomycin was bactericidal against MRSA 33591 ata concentration of 4 μg/mL in 24 hours.

Example 18 In Vivo Efficacy Studies in Neutropenic Mice

Animals (Male CD-1 mice, 20-30 g) were acquired from Charles RiversLaboratories (Gilroy, Calif.) and allowed access to food and water adlibitum. Neutropenia was induced via 200 mg/kg intraperitoneal (IP)injection of cyclophosphamide given four and two days prior to theinoculation of bacteria.

The organism used was either a susceptible or resistant strain ofclinically relevant Gram-positive pathogens, such asmethicillin-susceptible Staphylococcus aureus (MSSA 13709) andmethicillin-resistant Staphylococcus aureus (MRSA 33591). The bacterialinoculum concentration was ˜10⁶ CFU/mL. Animals were lightlyanesthetized with isoflurane and 50 mL of the bacterial inoculum wasinjected into the anterior thigh. One hour after the inoculation,animals were dosed intravenously with vehicle or the appropriate dose ofthe test compound. At 0 hours and 24 hours post-treatment, the animalswere euthanized (CO₂ asphyxiation) and the anterior and posterior thighcollected aseptically. The thigh was placed into 10 mL sterile salineand homogenized. Dilutions of the homogenate were plated onto tripticsoy agar plates which were incubated overnight. The number of bacterialcolonies on a given plate was multiplied by the dilution factor, dividedby the thigh weight (in grams) and expressed as log CFU/g. ED₅₀ (doserequired to produce 50% of the maximum reduction in thigh titre) wasestimated for each test compound.

In this assay, a compound of formula I, i.e. compound Ib, had an ED₅₀ of<0.1 mg/kg, iv, compared to an ED₅₀ of 9 mg/kg, iv, for vancomycin.

While the present invention has been described with reference tospecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited herein are incorporated by reference herein intheir entirety to the same extent as if they had been individuallyincorporated by reference.

1. A compound of the formula:

or a salt thereof, wherein: R² is hydrogen or C₁₋₆ alkyl; and R⁹ isselected from hydrogen, C₁₋₆ alkyl, and C₃₋₆ cycloalkyl, where the alkyland cycloalkyl groups are unsubstituted or substituted with —COOH or 1to 3 fluorine atoms.
 2. The compound of claim 1, wherein R⁹ is hydrogenor C₁₋₄ alkyl.
 3. The compound of claim 1, wherein R⁹ is hydrogen ormethyl.
 4. The compound of claim 1, wherein R² is hydrogen.
 5. Thecompound of claim 1, wherein R⁹ is methyl; and R² is hydrogen.
 6. Thecompound of claim 1, wherein R⁹ is methyl; and R² is ethyl.
 7. Acompound of the formula:

or a salt thereof, wherein: R² is hydrogen or C₁₋₆ alkyl; and R⁹ isselected from hydrogen, C₁₋₆ alkyl, and C₃₋₆ cycloalkyl, where the alkyland cycloalkyl groups are unsubstituted or substituted with —COOH or 1to 3 fluorine atoms.
 8. The compound of claim 7, wherein R⁹ is hydrogenor C₁₋₄ alkyl.
 9. The compound of claim 7, wherein R⁹ is hydrogen ormethyl.
 10. The compound of claim 7, wherein R² is hydrogen.
 11. Thecompound of claim 7, wherein R⁹ is methyl; and R² is hydrogen.